Imprinting is an epigenetic mechanism leading to mono-allelic expression of imprinted genes. In order to inherit the differential epigenetic imprints from one generation to the next, these imprints have to be erased in the primordial germ cells and re-established in a sex-specific manner during gametogenesis. The exact timing of the imprint resetting is not yet known and the use of immature gametes in assisted reproductive technologies may therefore lead to abnormal imprinting and related diseases. Imprinting is associated with differential allelic methylation in a CpG-context. We studied the methylation patterns of the imprint control (IC) region of the human SNRPN-gene in human spermatozoa, oocytes in different developmental stages [germinal vesicle (GV), metaphase I and metaphase II oocytes] and in preimplantation embryos using the bisulphite sequencing technique. In the spermatozoa, almost all potential methylation sites were unmethylated whereas mainly methylated patterns were found in the oocytes at different developmental stages. In the embryos, an average methylation pattern of 53% was found indicating that the imprints, which have been set during gametogenesis, are stably maintained in the preimplantation embryo. Our results indicate that the maternal imprints for the IC-region of the human SNRPN-gene are already re-established at the GV stage and that they are not re-established in a late oocyte stage or after fertilization as previously reported. Recent advances in assisted reproductive technologies raise questions concerning safety and the epigenetic risks involved. Our study was the first to check the methylation imprints in human pre-implantation embryos and oocytes at different developmental stages.
Imprinting is a non-Mendelian form of inheritance where epigenetic modifications control mono-allelic expression depending on the parental origin. Methylation of CpG-dinucleotides at differentially methylated regions (DMRs) is one of the best-studied mechanisms directing expression to one specific parental allele. We studied the methylation patterns of the intergenic (IG)-DMR of DLK1 and GTL2. The methylation marks of the IG-DMR were analysed in human gametes, preimplantation embryos, amniocytes and blood of babies born after intracytoplasmic sperm injection (ICSI) and blood from adults using a bisulphite sequencing technique. In oocytes, the IG-DMR was mainly unmethylated while in sperm cells a generally methylated pattern was detected. This germ-line specific methylation mark was maintained in the preimplantation embryos until the second cleavage stage. Afterwards in the preimplantation embryos, intermediate methylation patterns (26 -74% methylation) occurred, which may point to relaxation of the imprints. Intermediate patterns were also present in amniocytes, blood from ICSI babies and adults. We hypothesise that in the early cleavage stage embryo a strict differential methylation pattern is needed for the correct imprint establishment of surrounding imprinted genes. Once correct imprinting of the involved gene(s) is acquired, a more relaxed state of the IG-region is allowed.
Recently, several reports have been published that showed a higher incidence of assisted reproductive technologies (ART) in patients with Beckwith-Wiedemann syndrome compared with the general population, and in most of these patients, aberrant methylation imprints of KvDMR1 have been found. This has led to the concern that ART might increase the incidence of imprinting syndromes such as Beckwith-Wiedemann syndrome. Not much is known on environmental or genetic factors that may interfere with the processes of imprint maintenance or resetting. A methylation analysis of KvDMR1 was performed in human oocytes at different stages of nuclear maturity and in sperm cells. The results indicate that the maternal methylation imprints were already established at the germinal vesicle stage, whereas all sperm cells were unmethylated, thereby showing that the KvDMR1 carries a germline methylation imprint. For one of the oocytes analysed, an unmethylated pattern was found, which highlights the need for further molecular studies that consider the safety of ART.
BRCA1 and BRCA2 are the two primary breast cancer susceptibility genes in which identifying mutations is important to access cancer risk and decision for treatment. Prescreening of exons by High Resolution Melt (HRM) prior to sequencing can reduce effort in mutation screening in large transcripts such as the BRCA genes. Published methods for BRCA1 and 2 HRM screening have relied on complicated touchdown cycling protocols and custom reagent mixes that can lead to variable result. We have validated a streamlined workflow for adaptability in a clinical diagnostic environment. First, genomic DNA template was extracted on an automated sample processor using magnetic bead technology to purify genomic DNA directly from blood sample. Then, the PCR was performed using a PCR master mix optimized for HRM coupled with universal cycling conditions. We have successfully amplified all 120 amplicons covering exons of both BRCA genes using universal PCR condition. Potential variations identified by HRM screening were confirmed by direct sequencing of HRM PCR amplicons with M13 universal tags. By using unified protocol for PCR and universal sequencing for all amplicons the risk of incorrect procedure is greatly reduced. This simplified workflow for oncogene mutation screening using automated sample extraction and universal protocol for PCR and sequencing provides significant advantages for application in diagnostic settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2187.
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